Processing the 3′ end of a nascent transcript is critical for termination of RNA polymerase and for ensuring the proper functionality of the mature RNA. During normal development and in the progression of diseases such as cancer, 3′ end cleavage site usage frequently changes, resulting in additional sequence motifs being included (or excluded) at the 3′ ends of mature RNAs that can affect the transcripts' stability, subcellular localization, or function (reviewed in Lutz and Moreira 2011). Virtually all long RNA polymerase II (Pol II) transcripts terminate in a poly-A tail that is generated by endonucleolytic cleavage followed by the addition of adenosine (A) residues in a non-templated fashion (Moore and Sharp 1985; reviewed in Colgan and Manley 1997; Zhao et al. 1999; Proudfoot 2004). However, recent large-scale studies of the human transcriptome indicate that transcription is pervasive throughout the genome (reviewed in Wilusz et al. 2009) and suggest that a significant fraction (possibly >25%) of long Pol II transcripts present in cells may lack a canonical poly-A tail (Cheng et al. 2005; Wu et al. 2008; Yang et al. 2011a). Although some of these transcripts are likely degradation intermediates, there are well-characterized stable Pol II transcripts that lack a poly-A tail, such as replication-dependent histone mRNAs. Following U7 snRNA guided endonucleolytic cleavage at their 3′ end, histone mRNAs have a highly conserved stem-loop structure in their 3′ untranslated regions (UTRs) that is functionally analogous to a poly-A tail as it ensures RNA stability and enhances translational efficiency (reviewed in Marzluff et al. 2008).
Recent work has identified additional Pol II transcripts that are subjected to noncanonical 3′ end processing mechanisms (reviewed in Wilusz and Spector 2010). In particular, enzymes with well-known roles in other RNA processing events, such as pre-mRNA splicing (Box et al. 2008) and tRNA biogenesis, have been shown to cleave certain nascent transcripts to generate mature 3′ ends. In its well-characterized role, RNase P endonucleolytically cleaves tRNA precursors to produce the mature 5′ termini of functional tRNAs (reviewed in Kirsebom 2007). It was shown that RNase P also generates the mature 3′ end of the long noncoding RNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), also known as NEAT2, despite the presence of a nearby polyadenylation signal (Wilusz et al. 2008). Cleavage by RNase P simultaneously generates the mature 3′ end of the ˜6.7-kb MALAT1 noncoding RNA and the 5′ end of a small tRNA-like transcript. Additional enzymes involved in tRNA biogenesis, including RNase Z and the CCA-adding enzyme, further process the small RNA to generate the mature 61-nucleotide (nt) transcript known as mascRNA (MALAT1-associated small cytoplasmic RNA) (Wilusz et al. 2008).
The long MALAT1 transcript is retained in the nucleus in nuclear speckles (Hutchinson et al. 2007), where it has been proposed to regulate alternative splicing (Tripathi et al. 2010), transcriptional activation (Yang et al. 2011b), and the expression of nearby genes in cis (Nakagawa et al. 2012; Zhang et al. 2012). Although the MALAT1 locus appears to be dispensable for mouse development (Eissmann et al. 2012; Nakagawa et al. 2012; Zhang et al. 2012), MALAT1 is over-expressed in many human cancers (Ji et al. 2003; Lin et al. 2007; Lai et al. 2011), suggesting it may have an important function during cancer progression. Further, chromosomal translocation breakpoints (Davis et al. 2003; Kuiper et al. 2003; Rajaram et al. 2007) as well as point mutations and short deletions (Ellis et al. 2012) associated with cancer have been identified within MALAT1.
Despite lacking a canonical poly-A tail, MALAT1 is among the most abundant long noncoding RNAs in mouse and human cells. In fact, MALAT1 is expressed at a level comparable or higher than many protein-coding genes, including β-actin or GAPDH (Zhang et al. 2012).